The invention relates to the field of detection of proteins in biological samples. More specifically, the invention relates to methods and kits for detecting proteins and prions in a sample containing biological substances, and compositions employed by those methods and kits.
Numerous diseases and disorders affecting humans and animals have been determined or postulated to involve misfolded cellular proteins. Misfolding of naturally occurring normal cellular proteins is thought to cause the protein to lose activity, or, in many cases, behave abnormally. Misfolding of normal cellular proteins often causes formation of high molecular weight deposits or plaques within affected cells. Diseases that are known or thought to be associated with such misfolding of normal cellular proteins include Alzheimer's disease (AD), in which the A beta (amyloid beta) protein is involved; cerebral amyloid angiopathy (CAA); Parkinson's Disease, in which α-synuclein deposits in Lewy bodies are involved; Pick's Disease and frontal temporal dementia, in which the tau protein is involved; Amylotrophic Lateral Sclerosis (AML), in which superoxide dismutase is involved; Huntington's Disease, in which the protein huntingtin is involved; and the numerous Transmissible Spongiform Encephalopathies (TSE), such as Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Bovine Spongiform Encephalopathy (BSE; Mad Cow Disease), in which prion proteins are involved.
Many of the diseases and disorders associated with misfolded proteins and the associated deposits and plaques of these proteins are diseases and disorders of the central nervous system. Indeed, most of the diseases and disorders are neurodegenerative in nature, causing diminished neural cell function or neural cell death. The mechanisms by which deposits or plaques are formed from the misfolded proteins, and the relationship of deposit or plaque formation to the disease-associated neurodegenerative processes are not well-defined.
A group of proteins that are among these plaque-forming proteins are the prion proteins. Currently, prion proteins are under intense investigation due to their association with neurodegenerative diseases in farm animals and the human population, and their apparent transmission through biological materials from one animal or human to another, including apparent cross-species transmission.
It is widely held that prions, which are the infections agents of neurodegenerative diseases, are, in fact, entirely proteinaceous, and it has been postulated that the prion protein is the entity known as a prion. The prion protein is a protein referred to as PrP 27-30 or PrPC. It is an approximately 28-kilodalton hydrophobic glycoprotein that, when misfolded, aggregates into rod-like filaments found as plaques or deposits in infected brains. Prions exist normally as innocuous cellular proteins. However, prions possess an innate capacity to convert their structure and cause several deadly brain diseases of the dementia type in humans and animals. The dominating hypothesis is that, unlike all other infectious pathogens, infection is caused by an abnormal conformation of the prion protein, which acts as a template and converts normal prion conformations into abnormal conformations.
Complete prion protein-encoding genes have been cloned, sequenced, and expressed in transgenic animals. PrPC is encoded by a single-copy cellular gene and is normally found at the outer surface of neurons, attached to the membrane by way of a glycolipid moiety at the C-terminal portion of the protein. Cycling of the protein from the surface to the interior is known to occur, but the function of this cycling is not yet elucidated. Through an as-yet unclear post-translational process, under certain circumstances, PrPSc is formed from the normal, cellular PrPC. Neurodegenerative disease then can develop, a process that can take as much as decades to become clinically evident in humans.
The delay between the time of infection and manifestation of clinical symptoms of neurodegenerative disease is cause for heightened concern because this delay can permit unknowing transmission of the disease from one individual to others. For example, it can permit an animal infected with a prion to be slaughtered, and the potentially infectious products from that animal to enter the animal or human food stream. Likewise, it can permit an infected human to transmit infectious material to others through blood or organ donation. Thus, it is of great interest to the health community, the farming industry, and the general public to monitor animals and animal products (such as meat), and to monitor medical samples derived from humans (such as blood donations and organ donations) to reduce or eliminate the likelihood of transmission of prion proteins from infected individuals to others.
The normal cellular protein (PrPC) can be differentiated from the misfolded, infective protein (PrPSc) by numerous methods, the most common of which being susceptibility or resistance to degradation by proteases, such as Proteinase K, and detection with antibodies raised against one form of the protein or the other, or prior to or after proteinase digestion.
Many of the current techniques used to detect the presence of prion-related infections rely on gross morphological changes in the brain, or immunochemical techniques that are generally applied only after clinical symptoms are manifest, and which require biopsy material or material taken post mortem. Of course, by the time gross morphological changes in the brain are evident, a human may have transmitted infections particles to others through blood donations or donation of organs or tissues. Similarly, by the time post mortem analysis of animal tissue has been completed, products from the animal may have already entered the food stream and potentially infected other animals or even humans.
Other techniques in development or use rely on catalytic propagation of the misfolded form of cellular proteins involved in neurodegenerative diseases, such as prion protein. For example, Published U.S. Patent Application 2005/0026165 A1, which is incorporated herein in its entirety by reference, discloses the use of peptide probes to detect small amounts of misfolded protein in samples. In 2005/0026165 A1, peptides having sequences that bind to misfolded proteins are exposed to samples suspected of containing the misfolded protein. If misfolded protein is present, the probes bind to the misfolded protein. Binding causes a conformational change in the probe that permits it to bind to other probe molecules. Binding of other probe molecules to the misfolded protein-probe complex causes a conformational change in the newly bound probe, which converts it to a conformation that is capable of binding to yet another probe molecule in the mixture. The design of the probes permits a detectable signal to be generated when the target misfolded protein is present.
Although numerous techniques for detecting misfolded proteins, such as infectious prion proteins, exist in the art, there still exists a need for improved techniques that are rapid, inexpensive, reliable, reproducible, easy to use, and/or provide improved sensitivity. Improving the sensitivity relates to virtually all other needs. Furthermore, there exists a need for this technique to be convenient to carry out by means of a high-throughput kit.